The present invention generally relates to control systems and apparatuses for use with laser excitation or ionization, and more particularly to a system and apparatus which employs a laser, pulse shaper, mass spectrometer and electrical control system.
Conventionally, laser desorption mass spectrometry has been used with a fixed laser beam pulse shape and computers for simple chemical analysis processes on purified molecules with or without a matrix. The laser beam pulse shape was not considered an important parameter and was not modified; whatever fixed shape was set by the manufacturer for the ultraviolet laser was used in the tests. The general concept of typically laser selective ion formation from molecules in a molecular beam is disclosed in the following publication: Assion et al., “Control of Chemical Reactions by Feedback-Optimized Phase-Shaped Femtosecond Laser Pulses,” Science, Vol. 282, page 919 (Oct. 30, 1998). The pulse shaping process with a learning algorithm is disclosed in Judson et al., “Teaching Lasers to Control Molecules,” Physical Review Letters, Vol. 68, No. 10, page 1500 (Mar. 9, 1992). It is noteworthy, however, that the Assion article discloses use of an 80 femtosecond laser pulse and requires molecules to be isolated in a molecular beam, while the Judson article discloses use of a one nanosecond laser pulse and is purely conceptual as it does not include experimental results.
It is also known to employ nanosecond lasers for matrix-assisted laser desorption ionization (hereinafter “MALDI”). Examples of this are disclosed in U.S. Pat. No. 6,130,426 entitled “Kinetic Energy Focusing for Pulsed Ion Desorption Mass Spectrometry” which issued to Laukien et al. on Oct. 10, 2000, and U.S. Pat. No. 6,111,251 entitled “Method and Apparatus for MALDI Analysis” which issued to Hillenkamp on Aug. 29, 2000; both of these patents are incorporated by reference herein.
Until recently, commercially practical femtosecond lasers have been unavailable. For example, lasers which can generate 10 femtosecond or less laser pulse durations have traditionally been extremely expensive, required unrealistically high electrical energy consumption (for extensive cooling, by way of example) and depended on laser dyes that had to be replenished every month thereby leading to commercial impracticality. The efficiency of sub-10 femtosecond lasers was not practical until the year 2000 because of the prior need for dyes and flash lamps instead of YAG and Ti: Sapphire crystals pumped by light or laser emitting diodes.
Furthermore, the traditional role of the laser in a mass spectrometer with MALDI is to provide energy to the matrix molecules, wherein this energy dissipates and causes evaporation and ionization of the protein analyte dissolved in it. The laser, therefore, plays an indirect role that depends on energy transfer processes that may take from picoseconds to microseconds. Because excitation is indirect, pulse wavelength has not been found to cause significant differences in the outcome. Direct laser excitation of the proteins with nanosecond lasers typically causes the proteins to char.
In contrast, the present invention uses a different approach to MALDI in which the laser plays a more active and direct role in the ionization and even selective fragmentation of the analyte proteins. Shaped femtosecond pulses are required to achieve this goal. The optimum pulse shape cannot be found using the traditional laser sources, and trial and error. This is because the search for an optimal laser pulse shape involves a very wide range of possibilities. For example, if a 100 femtosecond laser pulse is used to produce pulse trains as long as several picoseconds in duration, splitting the laser beam spectrum into at least 100 spectral components is required since the length of the pulse is roughly inversely proportional to the band width. Since each component can be attenuated in 10 steps or phases shifted over 10 angles, then there are (10×10)100 different possible pulse shapes, and it would be impractical to systemically explore even a subset of these pulse shapes through conventional trial and error methods.
Laser induced, selective chemical bond cleavage has also been explored but with fairly limited success. It is believed that very simple molecules, such a HOD (partially deuterated water), have had only the OH and OD bonds cleaved with a nanosecond narrow line laser to vibrationally excite the specimen and then an ultraviolet laser pulse was employed to perform the cleaving. The desired laser frequency for vibrational excitation could be determined a priori in the gas-phase sample. More importantly, the HOD molecule is unique because the energy can be deposited in one of the bonds and it remains there for very long times, which are longer than nanoseconds. For the HOD experiments using selective bond excitation, no appreciable pulse shaping was used. This method was not known to have been employed for a protein or MALDI process, and was not known to have been successfully used for any other atomic bonds in other molecules, especially not in a condensed phase. It is also noteworthy that MALDI, with a matrix, has been used in an attempt to perform limited bond cleavage, as is discussed in U.S. Pat. No. 6,156,527 entitled “Characterizing Polypeptides” which issued to Schmidt et al. on Dec. 5, 2000, and is incorporated by reference herein. However, the approach of Schmidt et al. does not modify and optimize the laser pulse shape or other laser properties to achieve limited bond cleavage.
In accordance with the present invention, a control system and apparatus for use with laser excitation or ionization is provided. In another aspect of the present invention, the apparatus includes a laser, pulse shaper, detection device and control system. A further aspect of the present invention employs a femtosecond laser and a mass spectrometer. In yet another aspect of the present invention, the control system and apparatus are used in a MALDI process. Still another aspect of the present invention employs the control system and apparatus to cleave chemical bonds in a specimen and/or to determine the amino acid sequence of a protein specimen. Photodynamic therapy and fiber optic communication systems use the laser excitation apparatus with additional aspects of the present invention. A method of ionizing and determining a characteristic of a specimen is also provided.
The control system and apparatus of the present invention are advantageous over conventional constructions since the present invention allows for analysis and identification of constituents of complex and unknown molecules, such as those used in a MALDI process or proteins, in a relatively quick and automated manner. The present invention advantageously determines optimum laser conditions for maximizing the sensitivity of MALDI based protein sequencing, and to examine ion formation efficiencies for various matrices using tailored laser pulses. The present invention is also advantageously used to control the degree and type of fragmentation for automated protein sequencing. Furthermore, the adaptive laser source permits the optimal desorption from an insoluble protein source and allows for ionization analysis of a protein with or without a matrix.
The present invention is advantageous by employing ultra-fast laser beam pulses which can be repeatedly transmitted onto a specimen at least 1,000 times without replacing the specimen and without significant degradation of results. The ultra-fast laser also does not over-heat or “cook” a specimen, such as a protein. Recent improvements and efficiencies of femtosecond lasers have allowed for their commercially practical usefulness with the present invention. The automated feedback and pulse shaping of the control system of the present invention enhances signal-to-background sensitivity, especially for MALDI-based protein sequencing, while also statistically optimizing the process; this leads to significant time, cost and accuracy improvements. Additional advantages and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.